Kerstin Bluhm

N-Cyano Sulfoximines: COX Inhibition, Anticancer Activity, Cellular Toxicity, and Mutagenicity

From insects to cancer: N-Cyano sulfoximines were evaluated for COX inhibition and antiproliferative activity against a panel of cancer cell lines. The most active compound exhibited potent COX-2 inhibition, some selectivity for COX-2 over COX-1, only slight cytotoxicity towards healthy cells (HaCaT skin cells), and no mutagenic potential (as determined by an Ames assay).

International round‐robin study on the Ames fluctuation test

An international round‐robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate‐incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round‐robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9‐mix. Validity criteria (acceptable spontaneous and positive control‐induced mutation counts) were fulfilled by 92–100%, depending on the test conditions. A two‐step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine‐square‐root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the Williams test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%. Environ. Mol. Mutagen. 2012.

Quantitative assessment of the embryotoxic potential of NSO-heterocyclic compounds using zebrafish (Danio rerio)

Heterocyclic aromatic compounds (NSO-HET) have frequently been detected in the environment. Several studies have concluded that NSO-HET pose a threat to organisms in waters, sediments and soils. However, few publications are available assessing the ecotoxicology of NSO-HET. The present study aims to assess the embryo toxicity of heterocycles using Danio rerio. A combination of the Fish Embryo Toxicity Test and analytical quantification should aid to determine the hazard potential. Changes of the total concentrations due to sorption or volatility were quantified by GC/MS. Loss of compounds during the test was observed primarily for volatile or hydrophobic NSO-HET. The LC50 calculated with nominal concentrations underestimates the toxicity by a factor up to 16 (2 h), demonstrating that a chemical analysis for comparing nominal and measured concentrations is essential for such investigations.

Toxicological and ecotoxicological potencies of biofuels used for the transport sector—a literature review

In the past few years, the development and use of biofuels for the transport sector have attracted growing attention worldwide due to their promising benefits including a reduced dependence on fossil fuels and a potential to slow down the effect of global climate change. Nevertheless, concerns have also started to emerge regarding their potentially adverse environmental impacts and possible effects on human health. In this context, literature research was carried out to obtain an overview of the current research activities on the (eco)toxicological relevance of biofuels. The literature review revealed an increase in research activities on biofuels, in general, especially within the last four years. In contrast, comparatively few research activities were focused on the (eco)toxicological effectiveness of biofuels or their emissions even though this topic will be of great relevance as soon as a biofuel becomes commercially marketed in the future. Furthermore, the results of the available studies vary widely. Several findings on acute and mechanism-specific toxicity indicate less or comparable effects induced by biofuels in comparison to fossil diesel fuels. However, indications for negative impacts that are inducible both by the biofuels themselves and their emissions were found. Based on the data available, an (eco)toxicological relevance or human health risks associated with spills or the use of biofuels currently cannot be ruled out. Therefore, additional experimental studies are necessary to provide a more comprehensive dataset for the identification of future alternative fuels with low environmental impact.

Some heterocyclic aromatic compounds are Ah receptor agonists in the DR-CALUX assay and the EROD assay with RTL-W1 cells

PurposeHeterocyclic aromatic compounds containing nitrogen, sulfur, or oxygen heteroatoms (NSO-HET) have been detected in air, soil, marine, and freshwater systems. However, only few publications are available investigating NSO-HET using in vitro bioassays. To support better characterization of environmental samples, selected NSO-HET were screened for dioxin-like activity in two bioassays.MethodsThe present study focuses on the identification and quantification of dioxin-like effects of 12 NSO-HET using the DR-CALUX assay, and the 7-ethoxyresorufin-O-deethylase (EROD) assay with the permanent fish liver cell line RTL-W1. Changes of the total medium compound concentrations during the test procedure due to, e.g., sorption or volatilization were quantified using GC/MS.ResultsThe NSO-HET benzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophen, acridine, xanthene, and carbazole caused a response in the DR-CALUX assay. Only benzofuran and 2,3-dimethylbenzofuran were also positive in the EROD assay. All other compounds were inactive in the EROD assay. Relative potency (REP) values ranged from (2.80 ± 1.32) · 10−8 to (3.26 ± 2.03) · 10−6 in the DR-CALUX and from (3.26 ± 0.91) · 10−7 to (4.87 ± 1.97) · 10−7 in the EROD assay.ConclusionsThe REP values were comparable to those of larger polycyclic aromatic hydrocarbons, e.g., fluoranthene and pyrene. Thus, and because of the ubiquitous distribution of heterocyclic aromatic compounds in the environment, the provided data will further facilitate the bioanalytical and analytical characterization of environmental samples towards these toxicants.

A combined DNA-microarray and mechanism-specific toxicity approach with zebrafish embryos to investigate the pollution of river sediments

The zebrafish embryo has repeatedly proved to be a useful model for the analysis of effects by environmental toxicants. This proof-of-concept study was performed to investigate if an approach combining mechanism-specific bioassays with microarray techniques can obtain more in-depth insights into the ecotoxicity of complex pollutant mixtures as present, e.g., in sediment extracts. For this end, altered gene expression was compared to data from established bioassays as well as to results from chemical analysis. Mechanism-specific biotests indicated a defined hazard potential of the sediment extracts, and microarray analysis revealed several classes of significantly regulated genes which could be related to the hazard potential. Results indicate that potential classes of contaminants can be assigned to sediment extracts by both classical biomarker genes and corresponding expression profile analyses of known substances. However, it is difficult to distinguish between specific responses and more universal detoxification of the organism.

Heterocyclic Aromatic Hydrocarbons Show Estrogenic Activity upon Metabolization in a Recombinant Transactivation Assay

Heterocyclic aromatic hydrocarbons (hetero-PAHs) are increasingly studied at contaminated sites; especially at former industrial facilities where coal tar-oil was handled, e.g., wood treatment plants, high concentrations of hetero-PAHs are frequently detected in groundwater plumes. In previous studies, fractions of groundwater with high estrogenic activity contained hetero-PAHs and their hydroxylated metabolites. To evaluate this preliminary evidence, selected hetero-PAHs were screened for their estrogenic activity in lyticase yeast estrogen screen (LYES) and ER CALUX. All tested substances were inactive in the LYES. Hetero-PAHs such as acridine, xanthene, indole, 2-methylbenzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophene, quinoline, and 6-methylquinoline were positive in the ER CALUX, with estradiol equivalence factors (EEFs) from 2.85 × 10(-7) to 3.18 × 10(-5). The EEF values of these substances were comparable to those of other xenoestrogens (e.g., alkylphenols or bisphenol A) that are sometimes found in surface water. Chemical analyses revealed that T47Dluc cells could metabolize most of the substances. Among the metabolites (tentatively) identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were hydroxides and their keto tautomers, sulfates, sulfoxides, and N-oxides. Because of their high concentrations measured in groundwater, we conclude that hetero-PAHs and metabolites may be a potential risk and should be the subject of further research.

Biotests for hazard assessment of biofuel fermentation

To meet the increasing demand for energy, development of alternative and renewable energy sources, such as bioenergy, has accelerated during the last decade. In this context, biofuels are one potential replacement for fossil fuels, although their impact on the environment has not been widely studied. Only a few studies are available on toxicity of biofuels and biofuel combustion. Furthermore, for a complete understanding of the environmental impact, the entire life cycle of a biofuel has to be analyzed. This study is an exemplary ecotoxicological investigation of a biomass-to-biofuel production process with respect to the generation of environmentally relevant contaminants either by means of biomass pretreatment or microbial activity. Our aim is the demonstration of the suitability of ecotoxicological biotests as part of a comprehensive hazard assessment of biofuels and related samples or processes. Five ecotoxicological endpoints were assayed to determine the impact of four different biomass pretreatments on process substrates and effluent toxicities. Four different test organisms (bacterium, yeast, fish cell line, and fish embryo) from different trophic levels as well as a combination of acute and mechanism-specific biotests were applied to strengthen the ecotoxicological relevance of this investigation. Biotest results revealed cytotoxic, acute embryotoxic and mutagenic effectiveness, and weak estrogenic activity, with biomass toxicity depending on the mechanism of substrate pretreatment. Open microbial communities (reactor microbiomes) involved in the production process decreased the toxicity considerably to levels of the product n-butyric acid due to degradation of inhibiting by-products, verifying their simultaneous biomass conversion and detoxification potential. Our results demonstrate that ecotoxicological biotests are useful tools for the biofuel industries to gain environmental friendliness as a selling point.

Yangtze Three Gorges Reservoir, China: A holistic assessment of organic pollution, mutagenic effects of sediments and genotoxic impacts on fish

Besides obvious benefits, the Three Gorges Dams construction resulted in new pollution scenarios with the potentials to threaten the Three Gorges Reservoir (TGR) ecosystem. In order to record organic contamination, to find links to ecotoxicological impacts and to serve as reference for ensuing monitoring, several sites in the TGR area were screened applying the triad approach with additional lines-of-evidence as a holistic assessment method. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011 and 2012 to determine organic pollution levels, mutagenic potentials and genotoxic impacts. Two regional hot-spots near the cities of Chongqing and Kaixian were identified and further investigated in 2013. Only polycyclic aromatic hydrocarbons (PAHs) could be detected in sediments in 2011 (165-1653ng/g), emphasizing their roles as key pollutants of the area. Their ubiquity was confirmed at Chongqing (150-433ng/g) and Kaixian (127-590ng/g) in 2013. Concentrations were comparable to other major Chinese and German rivers. However, the immense sediment influx suggested a deposition of 216-636kgPAH/day (0.2-0.6mgPAH/(m(2)·day)), indicating an ecotoxicological risk. PAH source analysis highlighted primary impacts of combustion sources on the more industrialized upper TGR section, whereas petrogenic sources dominated the mid-low section. Furthermore, sediment extracts from several sites exhibited significant activities of frameshift promutagens in the Ames fluctuation assay. Additionally, significant genotoxic impairments in erythrocytes of P. vachellii were detected (Chongqing/Kaixian), demonstrating the relevance of genotoxicity as an important mode of action in the TGRs fish. PAHs, their derivatives and non-target compounds are considered as main causative agents.

Genotoxicity of Heterocyclic PAHs in the Micronucleus Assay with the Fish Liver Cell Line RTL-W1

Heterocyclic aromatic hydrocarbons are, together with their un-substituted analogues, widely distributed throughout all environmental compartments. While fate and effects of homocyclic PAHs are well-understood, there are still data gaps concerning the ecotoxicology of heterocyclic PAHs: Only few publications are available investigating these substances using in vitro bioassays. Here, we present a study focusing on the identification and quantification of clastogenic and aneugenic effects in the micronucleus assay with the fish liver cell line RTL-W1 that was originally derived from rainbow trout (Oncorhynchus mykiss). Real concentrations of the test items after incubation without cells were determined to assess chemical losses due to, e.g., sorption or volatilization, by means of gas chromatography-mass spectrometry. We were able to show genotoxic effects for six compounds that have not been reported in vertebrate systems before. Out of the tested substances, 2,3-dimethylbenzofuran, benzothiophene, quinoline and 6-methylquinoline did not cause substantial induction of micronuclei in the cell line. Acridine caused the highest absolute induction. Carbazole, acridine and dibenzothiophene were the most potent substances compared with 4-nitroquinoline oxide, a well characterized genotoxicant with high potency used as standard. Dibenzofuran was positive in our investigation and tested negative before in a mammalian system. Chemical losses during incubation ranged from 29.3% (acridine) to 91.7% (benzofuran) and may be a confounding factor in studies without chemical analyses, leading to an underestimation of the real potency. The relative potency of the investigated substances was high compared with their un-substituted PAH analogues, only the latter being typically monitored as priority or indicator pollutants. Hetero-PAHs are widely distributed in the environment and even more mobile, e.g. in ground water, than homocyclic PAHs due to the higher water solubility. We conclude that this substance class poses a high risk to water quality and should be included in international monitoring programs.